I have a rather strange question, but why are the preperations in order to make competent cells so different between chemical competent made cells and electroporated cells?
(I can see that you do not use CaCl2 when preparing the electrocomp cells, but why the need to centrifuge so many times with water and glycerol by example.)

and why do you always need an OD of 0.6 when starting the protocols?

And a last question: you need to do every step with cold substances, why do the cells need to be cold?

thanks

-lucilius-

lucilius on May 29 2010, 09:33 AM said:

Hallo all,

I have a rather strange question, but why are the preperations in order to make competent cells so different between chemical competent made cells and electroporated cells?
(I can see that you do not use CaCl2 when preparing the electrocomp cells, but why the need to centrifuge so many times with water and glycerol by example.)

The two protocols differ greatly because the mechanism which they use to move DNA into the cell are very different

Chemical competent protocol is based on the observation that bacteria will take up DNA fragments by endocytosis. It was further observed that DNA uptake was enhanced if the DNA was present in the form of Ca-DNA precipitate.

So the main aim of the chemical competent protocol was to sugar coat the bacteria cell with a fine precipitate of Ca-DNA.

Electro competent cells on the other hand works on the observation that the cell membrane has dielectric properties. The membrane is composed of a bilayer of phospholipids, whose charges heads all point outward, with their polar tails forming the center of the membrane. Thus there are two change surfaces separated by a insulating middle wall.

Now if a strong enough potential difference were placed across this membrane, the electric dipole moment on the phospholipid molecule can be sufficient to cause some phospholipids to realign with the electric field. Should a bacteria cell be perpendicular to the electric field, this realignment of phospholipid will cause the formation of holes in the cell wall. If electric field is not too high, these hole will be not too frequent and small, enabling the holes to spontaneously reseal themselves,

And remember DNA is changed, and thus moves in an electric field. So while these holes are present in the cell membrane, the DNA can be dragged by the electric field through the membrane into the cell. The cell membrane takes a little while to reseal, so DNA uptake continues (at a lower rate) for about 15min after electroporation

Thus the main aim of the electroporation protocol is to permeabilized the cell membrane, making it easier for phospholipid to move (and thus flip when exposed to an electric field).

lucilius on May 29 2010, 09:33 AM said:

and why do you always need an OD of 0.6 when starting the protocols?

Cell growing in mid log phase take up DNA better than cells at stationary phase. I do not know the actually reason for this, but it probably has to do with cell membrane and cell wall physiology. I do know that at mid log phase E coli cell wall has less polyglycans and liposaccarides in compared to stationary phase. And growing cells lyse more easier than stationary cells, probably indicating that there is less cell wall has less integrity.

lucilius on May 29 2010, 09:33 AM said:

And a last question: you need to do every step with cold substances, why do the cells need to be cold?

The competent cell protocol destabalises the cell wall and cell membrane, making it week and fragile. We keep the cell cool to stop this fragile cell from falling apart and leaking lots of cytoplasm.